Microarray analysis of Akt PH domain binding employing synthetic biotinylated analogs of all seven phosphoinositide headgroup isomers.

Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIP(n)s), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIP(n) binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein-PIP(n) binding interactions is hindered by the complexity of the membrane environment and of the PIP(n) structures. Herein, we describe two rapid assays of use for characterizing protein-PIP(n) binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIP(n) headgroup-biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P(3) and PI(3,4)P(2) as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIP(n) ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein-membrane binding interactions.

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P3-binding proteins.

Metabolically stabilized derivatives of phosphatidylinositol 4-phosphate: synthesis and applications.

Phosphatidylinositol 4-phosphate (PtdIns(4)P) lipid is an essential component of eukaryotic membranes and a marker of the Golgi complex. Here, we developed metabolically stabilized (ms) analogs of PtdIns(4)P and the inositol 1,4-bisphosphate (IP(2)) head group derivative and demonstrated that these compounds can substitute the natural lipid fully retaining its physiological activities. The methylenephosphonate (MP) and phosphorothioate (PT) analogs of PtdIns(4)P and the aminohexyl (AH)-IP(2) probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms analogs and IP(2) probes could be invaluable for identifying protein effectors and characterizing PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN).

Phosphatidylinositol 4,5-bisphosphate stimulates alveolar epithelial fluid clearance in male and female adult rats.

Cell membrane phospholipids, like phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], can regulate epithelial Na channel (ENaC) activity. Gender differences in lung ENaC expression have also been demonstrated. However, the effects in vivo on alveolar fluid clearance are uncertain. Thus PI(4,5)P(2) effects on alveolar fluid clearance were studied in male and female rats. An isosmolar 5% albumin solution was intrapulmonary instilled; alveolar fluid clearance was studied for 1 h. Female rats had a 37 ± 19% higher baseline alveolar fluid clearance than male rats. Bilateral ovariectomy attenuated this gender difference. Compared with controls, PI(4,5)P(2) instillation (300 µM) increased alveolar fluid clearance by ∼93% in both genders. Amiloride or the specific αENaC small-interfering RNA inhibited baseline and PI(4,5)P(2)-stimulated alveolar fluid clearance in both genders, indicating a dependence on amiloride-sensitive pathways. The fraction of amiloride inhibition was greater in PI(4,5)P(2)-instilled rats (male: 64 ± 10%; female: 70 ± 11%) than in controls (male: 30 ± 6%; female: 44 ± 8%). PI(4,5)P(2) instillation lacked additional alveolar fluid clearance stimulation above that of terbutaline, nor did propranolol inhibit alveolar fluid clearance after PI(4,5)P(2) instillation, indicating that PI(4,5)P(2) stimulation was not secondary to endogenous ß-adrenoceptor activation. PI(4,5)P(2) amine instillation resulted in an intermediate alveolar fluid clearance stimulation, suggesting that, to reach maximal alveolar fluid clearance stimulation, PI(4,5)P(2) must reside in cell membranes. In summary, PI(4,5)P(2) instillation upregulated in vivo alveolar fluid clearance similar to short-term ß-adrenoceptor upregulation of alveolar fluid clearance. PI(4,5)P(2) stimulation was mediated partly by increased amiloride-sensitive Na transport. There exist important gender-related effects suggesting a female advantage that may have clinical implications for resolution of acute lung injury.

Modular synthesis of biologically active phosphatidic acid probes using click chemistry.

Phosphatidic acid (PA) is an important signaling lipid that plays roles in a range of biological processes including both physiological and pathophysiological events. PA is one of a number of signaling lipids that can act as site-specific ligands for protein receptors in binding events that enforce membrane association and generally regulate both receptor function and subcellular localization. However, elucidation of the full scope of PA activities has proven problematic, primarily due to the lack of a consensus sequence among PA-binding receptors. Thus, experimental approaches, such as those employing lipid probes, are necessary for characterizing interactions at the molecular level. Herein, we describe an efficient modular approach to the synthesis of a range of PA probes that employs a late stage introduction of reporter groups. This strategy was exploited in the synthesis of PA probes bearing fluorescent and photoaffinity tags as well as a bifunctional probe containing both a photoaffinity moiety and an azide as a secondary handle for purification purposes. To discern the ability of these PA analogs to mimic the natural lipid in protein-binding properties, each compound was incorporated into vesicles for binding studies using a known PA receptor, the C2 domain of PKCalpha. In these studies, each compound exhibited binding properties that were comparable to those of synthetic PA, indicating their viability as probes for effectively studying the activities of PA in cellular processes.

Microplate-based characterization of protein-phosphoinositide binding interactions using a synthetic biotinylated headgroup analogue.

Membrane lipids act as important regulators of a litany of important physiological and pathophysiological events. Many of them act as site-specific ligands for cytosolic proteins in binding events that recruit receptors to the cell surface and control both protein function and subcellular localization. Phosphatidylinositol phosphates (PIP(n)s) are a family of signaling lipids that regulate numerous cellular processes by interacting with a myriad of protein binding modules. Characterization of PIP(n)-binding proteins has been hampered by the lack of a rapid and convenient quantitative assay. Herein, microplate-based detection is presented as an effective approach to characterizing protein-PIP(n) binding interactions at the molecular level. With this assay, the binding of proteins to isolated PIP(n) headgroups is detected with high sensitivity using a platform that is amenable to high-throughput screening. In the studies described herein, biotinylated PI-(4,5)-P(2) headgroup analogue 1 was designed, synthesized, and immobilized onto 96-well streptavidin-coated microplates to study receptor binding. This assay was used to characterize the binding of the PH domain of beta-spectrin to this headgroup. The high affinity interaction that was detected for surface association (K(d, surf) = 6 nM +/- 3), demonstrates that receptor binding modules can form high affinity interactions with lipid headgroups outside of a membrane environment. The results also indicate the feasibility of the assay for rapid characterization of PIP(n)-binding proteins as well as the promise for high-throughput analysis of protein-PIP(n) binding interactions. Finally, this assay was also employed to characterize the inhibition of the binding of receptors to the PIP(n)-derivatized microplates using solution phase competitors. This showcases the viability of this assay for rapid screening of inhibitors of PIP(n)-binding proteins.

Synthesis and convenient functionalization of azide-labeled diacylglycerol analogues for modular access to biologically active lipid probes.

Cell membrane lipids have been identified as key participants in cell signaling activities. One important role is their involvement as site-specific ligands in protein-membrane binding interactions, which result in the anchoring of peripheral proteins onto cellular membranes. These events generally regulate protein function and localization and have been implicated in both normal physiological processes and those pertaining to disease state onset. Thus, it is important to elucidate the details of interactions at the molecular level, such as lipid-binding specificities and affinities, the location of receptor binding domains and multivalency in binding. For this purpose, we have designed and developed azido-tagged lipid analogues as conveniently functionalizable lipid probe scaffolds. Herein, we report the design and synthesis of the initial structure of this type, diacylglycerol analogue 2, which contains an azide tag at the sn-1 position of the lipid headgroup. Direct functionalization of this compound with a range of reporter groups has been performed to illustrate the facile access to probes of use for characterizing binding. Quantitative lipid-binding studies using protein kinase C, a known DAG-binding receptor, demonstrate that these probes are active mimetics of natural DAG. Thus, these DAG probes will serve as robust sensors for studies aimed at understanding binding interactions and as precursors for the development of analogous probes of more complex phospholipids and glycolipids.